Methods for Reducing Cisplatin Nephrotoxicity

ABSTRACT

The present invention provides compositions and methods to reduce renal damage caused by nephrotoxic drugs such as cisplatin. The invention provides compositions comprising a substituted cyclodextrin, cisplatin and a pharmaceutically acceptable carrier, where the cyclodextrin is present in an amount effective for substantially inhibiting the nephrotoxic effect of the cisplatin.

This application is a continuation application of U.S. Ser. No.12/641,708 filed Dec. 18, 2009; which is a continuation application ofU.S. Ser. No. 11/753,883 filed May 25, 2007, now U.S. Pat. No.7,658,913, issued Feb. 9, 2010; which is a continuation-in-partapplication of U.S. Ser. No. 11/562,924 filed Nov. 22, 2006 and whichclaims the benefit of U.S. Provisional Application No. 60/740,142 filedNov. 28, 2005 and U.S. Provisional Application No. 60/778,037, filedMar. 1, 2006, the entire contents of each of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Numerous drugs and other substances are known to be nephrotoxic and cancause renal failure through a variety of mechanisms including directtoxicity to the renal tubules, allergic interstitial nephritis, andcrystallization of the drug within the renal tubules, which can lead toacute oliguric renal failure. Nephrotoxic drugs include anticanceragents such as cisplatin, methotrexate, and doxyrubicin, non-steroidalantiinflammatories (NSAIDS), such as COX-2 inhibitors, antibiotics(e.g., aminoglycosides, amphotericin) antivirals (e.g., acyclovir,indinivir), acetylcholinesterase inhibitors, angiotensin II receptorblockers (ARBs), lithium and radiographic contrast media.

A need exists to reduce renal damage caused by nephrotoxic drugs.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods to reduce renaldamage caused by nephrotoxic drugs. The invention provides compositionscomprising an anionically substituted oligosaccharide, a nephrotoxicdrug and a pharmaceutically acceptable carrier, where theoligosaccharide is present in an amount effective for substantiallyinhibiting the nephrotoxic effect of the drug.

Also provided are compositions having reduced nephrotoxic effectcomprising a pharmaceutically active compound having nephrotoxic effectand a polyanionic oligosaccharide where the oligosaccharide is presentin an amount effective to substantially reduce the nephrotoxic effect ofthe pharmaceutically active compound.

Also disclosed herein are methods of reducing the nephrotoxic effect ofa pharmaceutically active compound comprising contacting the compoundwith a polyanionic oligosaccharide. Additionally, methods are disclosedfor inhibiting nephrotoxicity associated with a nephrotoxic drug, themethod comprising administering a pharmaceutical composition comprisinga cyclic polysaccharide sulfate, the nephrotoxic inducing drug andoptionally a pharmaceutically acceptable carrier.

BRIEF SUMMARY OF THE FIGURES

FIG. 1 shows solubility study results with methotrexate (MTX) andcaptisol in aqueous acidic solution.

FIG. 2 shows renal pathology scores indicative of total kidney damage inmyelin-oligodendrocyte-glycoprotein (MOG) induced experimentalautoimmune encephalomyelitis (EAE) mouse model following treatment withMTX and MTX+captisol.

FIG. 3 shows clinical scores after treatment with MTX or MTX+captisol inEAE mice.

FIG. 4 shows renal pathology scores in kidney sections after singlebolus intravenous MTX with or without concurrent captisol at differentmolar ratios in normal mice.

FIG. 5 shows renal pathology scores in the kidney tissue of mice 24 and48 hours after treatment with MTX with or without concurrent captisol atdifferent molar ratios.

FIG. 6 shows the mean pathology scores in a doxorubicin inducednephrotoxic model for each treatment group at the level of superficialrenal cortex.

FIG. 7 shows the renal pathology scores for individual mice treated withdoxorubicin or doxorubicin+captisol at the level of superficial renalcortex.

FIG. 8 shows the mean pathology scores in a doxorubicin inducednephrotoxic model for each treatment group at the level of deep renalcortex+outer medulla.

FIG. 9 shows the renal pathology scores for individual mice treated withdoxorubicin or doxorubicin+captisol at the level of deep renalcortex+outer medulla.

FIG. 10 shows the mean scores at the level of the superficial cortex incisplatin and cisplatin+captisol treated groups.

FIG. 11 shows the pathology scores in a cisplatin induced nephrotoxicmodel of individual mice in each treatment group at the level of thesuperficial renal cortex.

FIG. 12 shows the mean scores at the level of the deep cortex and outermedulla in cisplatin and cisplatin+captisol treated groups.

FIG. 13 shows pathology scores in a cisplatin induced nephrotoxic modelof individual mice in each treatment group at the level of the deeprenal cortex and outer medulla.

FIG. 14 shows a comparison of the effect of cisplatin/HPβCD andcisplatin alone on the growth of leukemia cells.

DETAILED DESCRIPTION OF THE INVENTION

The compositions of the present invention typically comprise ananionically substituted oligosaccharide, a nephrotoxic drug andtypically a pharmaceutically acceptable carrier or other excipientcommonly used in the art. The oligosaccharide is present in an amounteffective to substantially inhibit the nephrotoxic effect of the drug.In one embodiment, the oligosaccharides are substituted with polar orcharged moieties, such as cationic or anionic substituents. In oneexample, the anionically substituted oligosaccharide is a polyanionicoligosaccharide comprising a cyclodextrin having one or more anionicsubstituents selected from the group consisting of sulfonate, sulfate,carboxylate, phosphonate and phosphate. In another embodiment, theoligosaccharide is a cyclic polysaccharide sulfate, preferably an α, βor γ-cyclodextrin sulfate.

The present invention also provides compositions having reducednephrotoxic effect comprising a pharmaceutically active compound havingnephrotoxic inducing effect and a polyanionic oligosaccharide.Nephrotoxic as used herein means toxic or destructive to the kidney, orany of its components.

Substituted Oligosaccharides

Substituted oligosaccharides generally refer to oligosaccharides havingat least one substituent per molecule, preferably a charged or polarsubstituent. The oligosaccharides are preferably saccharides of fromabout 5 to about 10 sugar units and have molecular weights, whenunsubstituted, from about 650 to about 1300. Where the oligosaccharideis anionically substituted, it is generally preferred that thesubstituents be selected from the group consisting of sulfonate,sulfate, carboxylate, phosphonate and phosphate groups and combinationsthereof. The substituents are preferably present in the molecule to anextent of from about 0.5 to about 3 substituents per sugar unit.Especially preferred compositions are those based on oligosaccharideshaving about 1 sulfonate substituent per sugar unit. Other preferredcompositions are based on oligosaccharides having from about 2 to about3 substituents per sugar unit, wherein the substituents comprisesulfate, sulfonate and/or phosphate substituents.

Oligosaccharides are chains of several sugar units such as glucoseunits, connected through glycosidic oxygen atoms. As used herein, theprefix “oligo” indicates an intermediate number of sugar or saccharideunits, as compared to a monomeric sugar unit of one, or at most two asin sucrose, and a polysaccharide having twenty or more of sugar unitsand high molecular weight. While all such oligosaccharides are believedto be operable within the scope of the present invention, theoligosaccharides hereof preferably have about 5 to about 10 saccharideunits per molecule. This range corresponds to unsubstituted saccharideshaving molecular weights ranging from about 650 to about 1300.Oligosaccharides having from about 5 to about 10 saccharide units permolecule are sometimes referred to herein as “simple” or “low molecularweight” oligosaccharides. Oligosaccharides are usually obtained byprocedures of degradation of starches or cellulose which result inoligosaccharide fragments in a broad range of sizes.

A somewhat related family of materials are the glycosaminoglycans. Theyare structures comprising a polysaccharide skeleton, modified by avariety of substituents containing nitrogen, sulfur and oxygen atoms,and comprising various segments such as glucosamines, iduronates,glucuronates and the like. Their structures are variable betweendifferent samples of the same name group, such as the chondroitans,dermatans, hyaluronic acid, heparan sulfates, and heparins. Each familyis known to be heterogenous, i.e., mixtures of compositions. Theirmolecular weight ranges generally between 10,000 and 25,000.

Substituted oligosaccharides, and, in particular, simple and lowmolecular weight oligosaccharides with polar or charged substituents,possess the ability to protect the kidneys from the nephrotoxic effectof certain classes of drugs. Anionically substituted cyclodextrins arepreferred, at least in part, because of the relative uniformity,improved solubility in aqueous solution such as the bloodstream,decreased toxicity, improved clearance from the body and ease ofproduction of such compounds, though other polar substituents such as OHmay be used.

Anionic substituents include, by way of example, those described in U.S.Pat. No. 3,426,011. Oligosaccharides may be of the general formula:

oligosaccharide-[(O—R—Y)⁻(Me)⁺]_(n)

where R is selected from the group consisting of straight chained orbranched C₁₋₁₀ alkyl, alkenyl or alkynyl; C₃₋₈ cycloalkyl and C₃₋₈ aryl,each ring optionally containing 1 or more heteroatoms selected from S, Nand O; and each of the aforementioned groups is optionally substitutedwith halo (i.e., F, Cl, Br, I) or hydroxyl;

Y is an acid group such as OH, COOH, SO₃, SO₄, PO₃H or PO₄, or aphosphorous, phosphinous, phosphonic, phosphinic, thiophosphonic,thiophosphinic and sulfonic acid; or is absent;

Me is a pharmaceutically acceptable anion or cation, such as lithium,sodium, potassium, calcium, magnesium, or aluminum, or an organicprimary, secondary, or tertiary amine such as methylamine,dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine,tributlyamine, pyridine, N,N-dimethylaniline, N-methylpiperidine,N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine,N,N-dibenzylphenethylamine, 1-ephenamine, andN,N′-dibenzylethylenediamine, ethylenediamine, ethanolamine,diethanolamine, piperidine, piperazine, and the like; and

n is the number of substituents per oligosaccharide, each of which isindependently selected, i.e., each substituent may be the same ordifferent. “N” will be a whole number greater than 1, the upper limitdependent upon the particular oligosaccharide. In a population ofoligosaccharides, it will be understood that n will represent theaverage number of substituents per molecule.

According to one embodiment, R is C₁₋₁₀ alkyl, preferably C₁₋₄ alkylselected from methyl, ethyl, propyl and butyl, each optionallysubstituted with halo or hydroxyl. Specifically preferred areoligosaccharides where in one or more groups, Y is SO₃. The resultingpreferred, polyanionically substituted oligosaccharides have molecularweights of from about 1600 to about 4000.

Cyclodextrins

In a preferred embodiment, the oligosaccharides are cyclicpolysaccharides, preferably cyclodextrins, and more preferablyderivatized cyclodextrins.

Cyclodextrins (also referred to as “CD” or “CDs”) are cyclicoligosaccharides consisting of at least six glucopyranose units.Although CDs with up to twelve glucopyranose units are known, only thefirst three homologs have been studied extensively, α, β, and γ, having6, 7 and 8 glucopyranose units, respectively. For example, theβ-cyclodextrin molecule is made up of seven α-1,4-linked glucopyranoseunits which form a cone-shaped molecule having a hydrophilic outersurface and a lipophilic cavity in the center. It is believed thatcyclodextrins exist as conical shaped molecules with the primaryhydroxyls situated at the small end of the cone and the secondaryhydroxyls situated at the large opening to the cone.

Topographically, the CDs may be represented as a torus, the upper rim ofwhich is lined with primary —CH₂OH groups, and the lower rim withsecondary hydroxyl groups. Coaxially aligned with the torus is achannel-like cavity of about 5, 6 or 7.5 A.U. diameter for the α, β, andγ-CDs, respectively. These cavities make the cyclodextrins capable offorming inclusion compounds with hydrophobic guest molecules of suitablediameters.

A reasonably large number of CD derivatives have been prepared anddescribed in the literature. In general, these chemically modified CDsare formed by reaction of the primary or secondary hydroxyl groupsattached to carbons 2, 3 or 6, without disturbing the α (1→4) hemiacetallinkages. A review of such preparations is given in Croft et al.,(Tetrahedron (1983) 39(9):1417-1474), incorporated herein by reference.Substitution via the hydroxyl groups on the glucopyranose units wouldinclude up to 18 for α-CD; 21 for (3-CD; and 24 for γ-CD. Thecyclodextrins may be selected from dextrins of the formula:

cyclodextrin-[(O—R—Y)⁻(Me)⁺]_(n)

where R, Y, Me and n are as described above. As will be apparent, n is 1to 18 for α-CD; 1 to 21 for (3-CD; and 1 to 24 for γ-CD.

Preferably, the cyclodextrin will have one or more substituents selectedfrom the group consisting of hydroxyl, sulfonate, sulfate, carboxylate,phosphonate and phosphate. According to one embodiment, R is straightchained or branched C₁₋₁₀ alkyl, preferably C₁₋₄ alkyl selected frommethyl, ethyl, propyl and butyl, each optionally substituted with haloor hydroxyl. Specifically preferred are oligosaccharides where in one ormore groups, Y is SO₃.

Preferred CDs are sulfate or sulfonate derivatives of α, β, andγ-cyclodextrins. Preparation of Cycloamylose Sulfates and Sulfonates,and Modified Cyclodextrin Sulfates and sulfonates are described in theart. See, for example, U.S. Pat. Nos. 2,923,704; 4,020,160; 4,247,535;4,258,180; 4,596,795 and 4,727,064, each of which is hereby incorporatedby reference. These cyclodextrin sulfates and sulfonates are typicallyassociated with a physiologically acceptable cation.

According to another embodiment, the hydroxyl groups are substitutedwith alkyl ether sulfonates of the formula —O—(C₁-C₈ alkyl)-SO₃. In oneexample, commercially available Captisol® (Cyclex) may be used which isa sulfobutyl ether derivative of (3-cyclodextrin having an average ofseven sulfobutyl ether groups per cyclodextrin molecule (i.e., O—R—Y is—O—(CH₂)₄—SO₃ ⁻Na⁺)(alternatively referred to herein as sulfobutyl ethylβ cyclodextrin or SBE-βCD). Captisol does not exhibit the nephrotoxicityassociated with underivitized β-cyclodextrin. Additional cyclodextrinderivatives are disclosed in U.S. Pat. Nos. 5,134,127; 6,165,995;6,479,467 (e.g., hydroxybutenyl-cyclodextrins); and 6,060,597, andpatent publication 20060258537 (SAE-AE-CD), each of which is herebyincorporated by reference. Additional CDs include methylated derivativeswith, for example, an average MS of 14 (M14-b-CD), and glucosyl andmaltosyl CDs containing mono-(G1-b-CD) and disaccharide (G2-b-CD)substituents. Additional cyclodextrins are set forth below (adapted fromMosher et al., Encyclopedia of Pharm. Tech. (2002) 531-58).

Nomenclature and Substituent Structures for Modified CDs

Position of Nomenclature #^(b)- substituent Substituent structure^(a)XYZ^(c)#^(d)-CD^(e) Parent cyclodextrins Alpha-CD —OH α-CD Beta-CD —OHβ-CD Gamma-CD —OH γ-CD Modified cyclodextrins neutral Methyl derivativesDimethyl 2,6- —O—CH₃ 2,6-DM14-CD Methyl Random —O—CH₃ M#-CD Trimethyl2,3,6- —O—CH₃ 2,3,6-TM-CD Ethyl derivatives Random —O—CH₂—CH₃ E#-CDHydroxyalkyl derivatives 2-hydroxyethyl Random —O—CH₂—CH₂OH (2HE)#-CD2-hydroxypropyl Random —O—CH₂—CHOH—CH₃ (2HP)#-CD or HP#-CD3-hydroxypropyl Random —O—CH₂—CH₂—CH₂OH (3HP)#-CD 2,3-dihydroxypropylRandom —O—CH₂—CHOH—CH₂OH (2,3-DHP)#-CD Modified cyclodextrins anionicCarbon Based Derivatives Carboxy 6- —CO₂M 6-C#-CD Carboxyalkyl Random—O—CH₂—CO₂M CM#-CD Carboxymethyl Random —O—CH₂—CH₂—CO₂M CE#-CDCarboxyethyl Random —O—CH₂—CH₂—CH₂—CO₂M CP#-CD Carboxypropyl 2,6-; 3-—O—CH₂—CO₂M; —O—CH₂—CH₃ CME#-CD Carboxylmethyl ethyl Sulfur BasedDerivatives Sulfates 2,6-random —O—SO₃M S#-CD Alkylsulfates 6-—O—(CH₂)11—O—SO₃M SU#-CD Sulfonates 6- —SO₃M 6-SA#-CD AlkylsulfonatesSulfoethyl ether Random —O—(CH₂)₂—SO₃M SEE#-CD Sulfopropyl ether Random—O—(CH₂)₃—SO₃M SPE#-CD Sulfobutyl ether Random —O—(CH₂)₄—SO₃M SBE#-CD^(a)M: Cation ^(b)Numbers represent position of substituents if known;if the preparation is a random distribution, then no notation implies anundefined distribution at the 2-, 3-, and 6- positions. ^(c)Lettersrepresent abbreviated notation of substituent. ^(d)Numbers represent theaverage MS rounded to the closest whole number. ^(e)Indication of parentCD structure, i.e., α-CD.

In another preferred embodiment, the cyclodextrin is of the formula:

cyclodextrin-[(O—R)⁻(Me)⁺]_(n)

where R is selected from the group consisting of straight-chained orbranched C₁₋₁₀ alkyl, alkenyl or alkynyl; substituted with 1 or morehydroxyl. In one embodiment, O—R is O—CH₂CH(OH)CH₃, i.e, thecyclodextrin is 2-hydroxypropyl β-cyclodextrin (HPβCD). In oneembodiment, the degree of substitution is 4.7, as used in the Examplesbelow.

The nephrotoxic drug may be any pharmaceutical agent including smallmolecules and peptides that cause renal damage upon administration to ahost. Such drugs include, by way of example, diuretics, NSAIDs, ACEinhibitors, cyclosporin, tacrolimus, radiocontrast media, interleukin-2,vasodilators (hydralazine, calcium-channel blockers, minoxidil,diazoxide), mitomycin C, conjugated estrogens, quinine, 5-fluorouracil,ticlopidine, clopidogrel, interferon, valacyclovir, gemcitabine,bleomycin, heparin, warfarin, streptokinase, aminoglycosides, cisplatin,nedaplatin, methoxyflurane, tetracycline, amphotericin B, cephaloridine,streptozocin, tacrolimus, carbamazepine, mithramycin, quinolones,foscarnet, pentamidine, intravenous gammaglobulin, fosfamide,zoledronate, cidofovir, adefovir, tenofovir, mannitol, dextran,hydroxyethylstarch, lovastatin, ethanol, codeine, barbiturates,diazepam, quinine, quinidine, sulfonamides, hydralazine, triamterene,nitrofurantoin, mephenyloin, penicillin, methicillin ampicillin,rifampin, sulfonamides, thiazides, cimetidine, phenyloin, allopurinol,cephalosporins, cytosine arabinoside, furosemide, interferon,ciprofloxacin, clarithromycin, telithromycin, rofecoxib, pantoprazole,omeprazole, atazanavir, gold, penicillamine, captopril, lithium,mefenamate, fenoprofen, mercury, interferon, pamidronate, fenclofenac,tolmetin, foscarnet, aciclovir, methotrexate, sulfanilamide,triamterene, indinavir, foscarnet, ganciclovir, methysergide,ergotamine, dihydroergotamine, methyldopa, pindolol, hydralazine,atenolol, taxol, tumor necrosis factor, chlorambucil, interleukins,bleomycin, etoposide, fluorouracil, vinblastine, doxorubicin, cisplatinand the like (see, generally, Devasmita et al., Nature Clinical PracticeNephrology (2006) 2, 80-91).

Methotrexate

According to one embodiment, the nephrotoxic drug is methotrexate, or aderivative or pharmaceutically acceptable salt thereof. Methotrexate(N-[4-[[(2,4-diamino-6pteridinyl)methyl]methylamino]benzoyl]-L-glutamicacid)is an S-phase chemotherapeutic antimetabolite used for the treatment ofvarious neoplasms, particularly CNS lymphoma. MTX is one the most widelyused anticancer agents and is employed in the treatment of neoplasticdiseases such as gestational choriocarcinoma, osteosarcoma,chorioadenoma destruens, hydatidiform mole, acute lymphocytic leukemia,breast cancer, epidermoid cancers of the head and neck, advanced mycosisfungoides, lung cancer, and non-Hodgkins lymphomas (Physicians DeskReference (45th ed.), Medical Economical Co., Inc., 1185-89 (Des Moines,Iowa (1991))). MTX is also an effective immunosuppressive agent, withutility in the prevention of the graft-versus-host reaction that canresult from tissue transplants, as well as in the management ofinflammatory diseases. Consequently, MTX can be employed in thetreatment of severe and disabling psoriasis and rheumatoid arthritis(Hoffmeister, The American Journal of Medicine (1983) 30:69-73; Jaffe,Arthritis and Rheumatism (1988) 31: 299).

However, methotrexate is associated with renal and hepatic toxicity whenapplied in the “high dose regimen” that is typically required formaximum efficiency (Barak et al., J. American Coll. Nutr. (1984)3:93-96).

Numerous patents disclose MTX and MTX analogs, any of which may be usedin practicing the present invention. See, for example, U.S. Pat. Nos.2,512,572, 3,892,801, 3,989,703, 4,057,548, 4,067,867, 4,079,056,4,080,325, 4,136,101, 4,224,446, 4,306,064, 4,374,987, 4,421,913,4,767,859, 3,981,983, 4,043,759, 4,093,607, 4,279,992, 4,376,767,4,401,592, 4,489,065, 4,622,218, 4,625,014, 4,638,045, 4,671,958,4,699,784, 4,785,080, 4,816,395, 4,886,780, 4,918,165, 4,925,662,4,939,240, 4,983,586, 4,997,913, 5,024,998, 5,028,697, 5,030,719,5,057,313, 5,059,413, 5,082,928, 5,106,950, 5,108,987, 4,106,488,4,558,690, 4,662,359, 6,559,149, each of which is hereby incorporated byreference. Other MTX analogs and related antifolate compounds includetrimetrexate, edatrexate, AG331, piritrexim, 1843U89, LY 231514, ZD9331, raltritrexed, lometrexol, MTA and AG337 (Takimoto, Seminars inOncology (1997) 24:S18-40-51; Sorbello et al., Haematoligica (2001)86:121-27); CB 3717, LY 309887 (Calvert, Seminars in Oncology (1999)26:S6, 3-10; Rosowsky, Progress in Med. Chem. (1989) 26:1-237)).

Accordingly, the compositions disclosed herein may be used for treatingcancer or for inhibiting the growth of cancer; as well as for treatingmultiple sclerosis, and the symptoms associated therewith. Thecompositions may be used in conjunction with or in combination withother active agents such as interferon. Additionally, the compositionsmay be used for treating autoimmune disorders such as lupus andrheumatoid arthritis.

Antibiotics

Aminoglycoside antibiotics, such as gentamicins, kanamycins,streptomycins and tobramycins, are generally utilized as broad spectrumantimicrobials effective against, for example, gram-positive,gram-negative and acid-fast bacteria. However, aminoglycosides are oftenassociated with undesired side-effects such as nephrotoxicity andototoxicity. Other antibiotics which may be used in the practice of thepresent invention include acyclovir, vancomicin and cephalosporin, suchas Rocephin® and Kefzol.

Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)

NSAIDs all present renal toxicity. NSAIDs typically are used to reducepain while avoiding the use of opiate derivatives. Two widely usedNSAIDs are Indomethacin and Toradol® manufactured by RochePharmaceuticals.

Antifungal Agents

Caspofungin and amphotericin B are both known to be nephrotoxic, and maybe used in practicing the disclosed invention.

Anti-Cancer Agents

Many anti-cancer agents exhibit dose limiting renal toxicity and may beused in practicing the present invention. Such agents include, by way ofexample, cisplatin, doxorubicin, cyclophosphamide, busulfan, and thelike.

Contrast Agents

Contrast agents are injected into a patient prior to x-ray scans.Contrast agents are highly concentrated (50-66% solutions) iodinatedcompounds. In view of this high concentration it is likely that only aminimum of a 1:1 ratio of contrast agent to cyclodextrin would benecessary, and up to about 10 to 1 or higher. Examples of the possibleuse of cyclodextrin to protect against renal damage by contrast agentsinclude Iohexyl and Ioversol. Other contrast agents that may be used inpracticing the present invention include diatrizoate meglumine andioxaglate.

Administration

The oligosaccharide may be complexed with the nephrotoxic drug, althoughit is not believed necessary for antinephrotoxic protective effect ofthe composition. The ratio of drug to oligosaccharide is preferablywithin a range such that the drug does not precipitate at pH typicallyfound in the kidney, taking into account the transit time of the drugthrough the kidney. In some cases, it may be desirable to minimize theamount of oligosaccharide in vivo. Simple in vitro solubilityexperiments such as described in the Examples may be used to determinethe minimum amount of oligosaccharide needed to effectively protect fromrenal damage. Alternatively, the minimum amount of oligosaccharideneeded to effectively protect the kidney from damage can be determinedin animal studies of the effect of the oligosaccharide on drug inducedkidney pathology.

In one embodiment, the molar ratio of drug:oligosaccharide is greaterthan 1:1, and may range from about 1.1:1 to about 50:1, preferably fromabout 1.25:1 to about 25:1; more preferably from about 1.75:1 to about2:1 to about 10:1. In the case of methotrexate, by way of example only,it was found that a mole ratio of about 2:1 methotrexate:Captisol workedwell to keep methotrexate in solution in vitro and provided the desirednephrotoxic effect. Where lower amounts of oligosaccharides are desired,it is contemplated that additional solubilizing agents may be used,provided that the amount of oligosaccharide in the composition remainssufficient to provide a renal protective effect. By way of example, inthe case of the contrast agent iohexyl, as shown in the Examples, a moleratio of about 10:1 iohexyl to SBEβCD worked well to markedly reduce thekidney pathology in iohexyl induced kidney damage in mice.

It may be desirable in some cases to have a ratio of drug tooligosaccharide lower than 1:1 where, for example, the binding constantof the drug is low or where the drug is processed by the kidneys at alower rate than the cyclodextrin, it may be beneficial to have a molarexcess of oligosaccharide. This may be true for many classes of drugswhere the dose of the drug required for a therapeutic effect is low,therefore, although the molar ratio of oligosaccharide might be higher,the absolute amount or concentration in vivo is not necessarilyincreased. As such, the composition may comprise from about 2 to about50; from about 2 to about 20; or from about 2 to about 10 times molarexcess of the oligosaccharide, or preferably in the range of from about1 to about 5:1, and more preferably in the range of about 2 to about5:1, oligosaccharide to drug.

Further provided are methods of reducing the nephrotoxic effect of apharmaceutically active compound comprising contacting the compound witha polyanionic oligosaccharide. Methods are included for inhibiting orreducing nephrotoxicity associated with a nephrotoxic drug, comprisingadministering a pharmaceutical composition comprising a polyanionicoligosaccharide, the nephrotoxic drug and optionally a pharmaceuticallyacceptable carrier. Although it is preferred that administration occuras a single dose, particularly where the oligosaccharide assists insolubilizing the drug, the methods may also be effected by concurrentlyadministering a pharmaceutical composition comprising a polyanionicoligosaccharide and a pharmaceutical composition comprising thenephrotoxic inducing drug, i.e., in separate doses. Where the drug andoligosaccharide are combined into a single dosage unit, they may becombined with a pharmaceutically acceptable carrier, for example, acosolution or dispersion in an inert pharmaceutically acceptable solventor dispersing agent or the like.

Alternatively, the oligosaccharide can be separately formulated withpharmaceutically acceptable materials and administered separately;either concurrently with the drug or within about an hour before orafter administration of the drug. By concurrently, it is meantadministration of the separate doses occurs substantially at the sametime such that both the oligosaccharide and the drug are present invivo. Alternatively, administration may occur sequentially provided thatthe oligosaccharides are present within the renal environment as theconcentration of drug in the kidney increases to levels at which thetoxic effect of the drug may occur.

The mode of administration, the dosage and frequency of dosage isgoverned by the mode of administration and dosage considerationsconventionally employed with the pharmaceutical agent. Thus, forexample, various combinations of the invention can be administeredintramuscularly or intravenously, or otherwise, as dictated by medicaland pharmacological practice related to the desired use of theparticular drug or agent employed. Administration may be achieved orallyor parenterally including, inter alia, topical application, intravenous,intra-arterial or subcutaneous injection, and including absorption aswell as injection and introduction into body apertures or orifices.

It will be understood that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy.

The other features of the invention will become apparent in the courseof the following descriptions of exemplary embodiments which are givenfor illustration of the invention and are not intended to be limitingthereof.

EXAMPLES Example 1 Effect of pH on Solubility of MTX

Solubility studies were performed to determine whether Captisol couldprevent the precipitation of MTX over a time period greater than transittime of MTX through the kidney (i.e., less than 2 minutes). Solutionswere prepared as shown in the Table 1. Each solution was acidified withHCl, centrifuged and aliquots of the supernatant removed as a functionof time, and the concentration of MTX in solution was measuredspectrophotometrically.

As shown in FIG. 1, Captisol prevented the precipitation of MTX at aconcentration dependent rate. At a mole ratio of 1:1, the MTX remains insolution indefinitely. At lower ratios, precipitation occurs at aconcentration dependent rate. At 0.50:1 ratio of Captisol to MTX, MTXremains in solution for at least 15 minutes, and at a ratio of 0.25:1,much of it remains for 10 minutes. In view of the rapid transit time offiltration in the kidneys, in vivo experiments to determine the optimalratio of MTX to Captisol to prevent kidney damage may be performed.

TABLE 1 MTX-captisol solubility MTX added: 0.91 mg/ml Concentration ofMTX: 0.002 M Rate of precipitation of MTX at pH 5.0 in presence andabsence of captisol Captisol:MTX molar ratio 1:1 Time after Amount ofcorrection for Captisol adding MTX dilution conc. of added acid OD at insolution 1:25 dilution MTX captisol/MTX (mM) (min) 302-304 nm (ug/mL)ug/ml mg/ml (M) ratio 0.002  5 2.71  33.28800989 832.2002 0.83220.0018308 1:1 0.002 10 2.291 28.10877627 702.7194 0.702719 0.001546 1:10.002 15 2.296 28.17058096 704.2645 0.704265 0.0015494 1:1 0.002 202.127 26.0815822 652.0396 0.65204 0.0014345 1:1 0.002 30 2.06725.33992583 633.4981 0.633498 0.0013937 1:1 0.002 40 2.202 27.00865266675.2163 0.675216 0.0014855 1:1 0.002 50 2.078 25.47589617 636.89740.636897 0.0014012 1:1 0.002 60 2.004 24.56118665 614.0297 0.614030.0013509 1:1 Captisol:MTX molar ratio 0.5:1 correction for Timedilution after 1:500 Captisol adding OD at MTX 1:25 conc. of added acid302-304 nm in solution dilution mtx captisol/MTX (M) (min) nm (ug/mL)ug/ml mg/ml (M) ratio 0.001  5 1.854 22.70704574 567.6761 0.5676760.0012489 0.5:1 0.001 10 2.263 27.76266996 694.0667 0.694067 0.00152690.5:1 0.001 15 2.594 31.85414091 796.3535 0.796354 0.001752 0.5:1 0.00120 1.531 18.7144623 467.8616 0.467862 0.0010293 0.5:1 0.001 30 1.04412.6946848 317.3671 0.317367 0.0006982 0.5:1 0.001 40 0.959 11.64400494291.1001 0.2911 0.0006404 0.5:1 0.001 50 0.864 10.4697157 261.74290.261743 0.0005758 0.5:1 0.001 60 0.861 10.43263288 260.8158 0.2608160.0005738 0.5:1 Captisol:MTX molar ratio 0.25:1 Time of OD min Captisolafter OD at Dilution molarity of added adding 302-304 nm conc of MTX1:500 mtx captisol/MTX (M) acid nm (ug/mL) ug/ml mg/ml (M) ratio 0.0005 5 2.363 28.99876391 724.9691 0.724969 0.0015949 0.25:1 0.0005 10 1.79822.01483313 550.3708 0.550371 0.0012108 0.25:1 0.0005 15 1.17314.28924598 357.2311 0.357231 0.0007859 0.25:1 0.0005 20 0.86410.4697157 261.7429 0.261743 0.0005758 0.25:1 0.0005 30 0.686 8.26946848206.7367 0.206737 0.0004548 0.25:1 0.0005 40 0.633 7.61433869 190.35850.190358 0.0004188 0.25:1 0.0005 50 0.556 6.662546354 166.5637 0.1665640.0003664 0.25:1 0.0005 60 0.556 6.662546354 166.5637 0.166564 0.00036640.25:1 Captisol:MTX molar ratio 0:1 Time of OD min Captisol after OD atdilution added adding 302-304 nm conc of MTX 1:500 molarity ofcaptisol/MTX (M) acid nm (ug/mL) ug/ml mg/ml mtx (M) ratio 0  5 0.2963.448702101 86.21755 0.086218 0.0001897 0:1 0 10 0.22  2.50927070562.73177 0.062732 0.000138 0:1 0 15 0.274 3.176761434 79.41904 0.0794190.0001747 0:1 0 20 0.328 3.844252163 96.1063 0.096106 0.0002114 0:1 0 300.325 3.807169345 95.17923 0.095179 0.0002094 0:1 0 40 0.284 3.30037082882.50927 0.082509 0.0001815 0:1 0 50 0.293 3.411619283 85.29048 0.085290.0001876 0:1 0 60 0.333 3.90605686 97.65142 0.097651 0.0002148 0:1

Example 2 Protective Effect of Captisol MTX

A comparison of the effect of MTX 40 mg/kg with and without Captisol(molar ratio 1:1) in myelin-oligodendrocyte-glycoprotein (MOG) inducedexperimental autoimmune encephalomyelitis (EAE) in C57BL6 mice wasperformed. Clinical signs, CNS pathology and renal pathology weremeasured.

EAE was induced as follows. Mice were anesthetized with Avertin (222tribromoethanol) and given two subcutaneous injections of 150 μg of MOGin PBS (total dose 300 μg) that had been emulsified in an equal volumeof Freund's incomplete adjuvant containing 250 μg of M. tuberculosisH₃₇RA (total dose 500 μg). One injection was given at the nape and thesecond was given on the dorsum. Pertussis toxin (100 ng; i.v. throughthe tail vein) was administered on days 0, 3, and 7 followingencephalitogen.

Stock MTX was used at 25 mg/ml (Bedford laboratories). MTX stock wasdiluted 3.67 times with PBS (2 ml stock+5.34 ml PBS) for a total volumeof 7.34 ml, at a concentration of 6.8 mg/ml (14.9 mM).

3.00 ml of diluted MTX solution was aliquoted and added to 96.6 mg ofCaptisol powder, and the solution vortex mixed. The resulting solutionwas mostly clear but pale yellow. All solutions were kept at RT in darkuntil ready to inject. 96.6 mg/3 ml=32.228 mg/m=14.9 mM; MTX: Captisolmolar ratio 1:1.

The test mixtures were administered to 5 groups of mice (Groups I-V; seeTable 2).

TABLE 2 Groups EAE/control Treatment Number of mice Group I EAE PBS 5Group II EAE MTX 40 mg/kg 10 Group III EAE MTX 40 mg/kg + 10 captisol(molar ratio 1:1) Group IV EAE Captisol only 10 201.25 mg/kg Group V EAENo treatment 5

Twenty four (24) hours after symptom onset, MTX (40 mg/kg body weight)was administered via tail vein. Injection volume were between 100-120μl. MTX+Captisol was injected in a volume of 100-120 μl via tail vein.Captisol alone (32.22 mg/ml) was injected via tail vein in a volume of100-1204 Leukovorin (20 mg/kg body weight) was injected via tail vein 4hours after MTX injection and again 24 hours after. Leukovorin, theactive metabolite of folic acid, is typically given with methotrexate inanti-cancer chemotherapy to help protect normal cells.

Animals were weighed and scored daily for clinical signs. Scores werebased on the following on the following signs:

0—normal

1—flacid tail, piloerection, and/or weight loss

2—hind limb weakness righting difficulty

3—hind limb weakness causing righting inability

4—hindlimb paresis, limp walking, and or/incontinence

5—partial hind limb paralysis

6—total hind limb paralysis plus forelimb weakness

7—total hind limb paralysis plus forelimb paresis or paralysis

8—death or moribund requiring sacrifice

Mice were scored daily for disease severity and then sacrificed on day10 of disease. Brain, kidney and spleen were formalin fixed. To assessthe infiltration of T cells into the CNS, CD3+immunohistochemistry wasperformed on paraffin embedded 8 micron thick hind brain sections ofuntreated EAE mice and EAE mice treated concurrently with MTX andcaptisol. For light microscopic investigation, kidneys were fixed in 10%buffered formalin and processed routinely for paraffin embedding. Tissuesections of 5 micron meter were stained with hematoxylin and eosine andexamined under Nikon coolpix light microscope.

The severity of total kidney damage was evaluated by a semiquantitativemeasurement of damage as described. Each tissue section of the kidneywas assessed for degeneration of glomerular structure, glomerularcrowding and congestion, dilation of bowman space, degeneration ofproximal and distal tubules, and dilation of renal tubules, vascularcongestion, and inflammatory cell infiltration. For glomerular atrophy,glomeruli that contained more than 20 nuclei were scored “0”, and thosecontaining less than 10 nuclei were scored “4.” Intermediate stages were1, 2 and 3. Other criterion was scored on a 0-3 scale: 0=none; 1=mild;2=moderate; 3=severe.

The microscopic score of each tissue was calculated as the sum of thescores given to each criterion and at least 100 nephrons (glomeruli plussurrounding tubules) were analyzed per section. Data is represented asmean±SEM (see FIG. 2).

MOG treated mice developed severe clinical manifestation, starting ondays 10-11. They were maintained until sacrifice at 13 days. All animalwere affected. Partial or complete hind leg paralysis (clinical score).

Efficacy of MTX 40 mg/kg+Captisol was comparable to the efficacy seenwith MTX treatment alone.

The result of the CNS Pathology—CD3′ Immunostaining showed thatuntreated EAE mice had extensive infiltration of CD3′ T cells in thehind brain and spinal cord (not shown). EAE mice treatedpost-symptomatically with concurrently MTX+Captisol (40 mg/kg+captisol1:1 molar ratio) showed 80-90% reduction in T cell infiltration (notshown). The efficacy of MTX+Captisol was comparable to the efficacy withMTX treatment alone.

Renal pathology scores are shown in FIG. 3. Kidney sections from 3different EAE mice treated with MTX 40 mg/kg, showed dilation of renaltubules and degeneration of proximal tubules. Kidney sections from micetreated concurrently with MTX+Captisol showed a protective effect ontubules. A single intravenous bolus injection of 40 mg/kg MTX producedmorphological changes in the kidney which was mostly restricted todilation of renal tubules in the cortex. Concurrent Captisoladministration with MTX resulted in reduction in pathology score in theEAE mice.

Example 3 Captisol MTX at Various Molar Ratios

A study of histopathological changes in the kidney after single bolusintravenous MTX with or without concurrent captisol at different molarratios was performed.

MTX solutions were prepared as follows. Solution A (24 mg/ml (53 mM))was prepared from stock solution of 25 mg/ml (Methotrexate from Bedfordlaboratories) in sterile PBS (total volume 6 mL). Solution B wasprepared by diluting 3 ml of Solution A to 1:1.33 in PBS to obtain aworking dilution of 18 mg/mL (39.6 mM), pH 7.4. Solution C was preparedby diluting 2 mL of solution A 1:2 to obtain a working dilution of 12mg/mL (26.4 mM), pH 7.2.

MTX+Captisol solutions were prepared as follows. Solution D was made byadding 57.32 mg Captisol to an aliquot of 500 micro liter of solution A(molar ratio 1:1, neutral pH). Solution E was made by adding 85.6 mgCaptisol to 1 mL aliquot of solution B (molar ratio 1:1, neutral pH).Solution F was made by adding 57.10 mg Captisol to 1 mL aliquot ofsolution C (molar ratio 1:1, neutral pH). Solution G was prepared byadding 42.8 mg Captisol to 1 mL aliquot of solution B. Solution H wasmade by adding 28.6 mg of Captisol to 1 mL aliquot of solution C.

MTX and Captisol were administered to 8 groups of mice (Groups I-VIII;see Table 3) via tail vein by a single injection in a volume of 100-120μL. Animals were sacrificed after 48 hours. Clinical symptoms, bodyweights were recorded. Kidneys were preserved (one frozen, one formalinfixed) for pathology. Leukovorin was given at 4 hours and after 18hours. Urine was not alkalinized.

TABLE 3 Molar Molarity of Molarity ratio # of GROUPS TREATMENT MTX ofCaptisol MTX:Captisol mice Group I Normal MTX only, 80 mg/kg, 26.4 mM 3mice iv bolus. Solution A Group II Normal MTX only, 120 mg/kg, 39.6 mM 3mice iv, bolus. Solution B Group Normal MTX only, 160 mg/kg,   53 mM 1III mice iv, bolus. Solution C Group Normal MTX, 80 mg/kg, iv + 26.4 mM26.4 mM 1:1 3 IV mice Captisol 382 mg/kg body weight. Solution D Group VNormal MTX, 120 mg/kg, iv + 39.6 mM 39.6 mM 1:1 3 mice Captisol 570mg/kg. Solution E Group Normal MTX, 160 mg/kg, iv +   53 mM   53 mM 1:11 VI mice Captisol 764 mg/kg body weight. Solution F Group Normal MTX,80 mg/kg, iv + 26.4 mM 13.2 mM   1:0.5 3 VII mice Captisol 191 mg/kg.Solution G Group Normal MTX, 120 mg/kg, iv + 39.6 mM 19.8 mM   1:0.5 3VIII mice Captisol 285 mg/kg. Solution H

Semi quantitative estimates of total kidney damage in mice treated withMTX 80 mg/kg, 120 mg/kg, and a combination of MTX+Captisol at MTX toCaptisol molar ratio of 1:1 and 1:0.5, are shown in FIG. 4. The scoresfor the control kidney were between 3-4 (not shown in the graph).Hematoxylin and eosin sections of paraffin embedded kidney were analyzedmicroscopically for extent of renal damage. Data is expressed asmean±SEM. Data for MTX 160 mg/kg is not included in the graph as therewas only a single mouse in this group.

Paraffin sections of the kidneys stained with hematoxylin and eosin (notshown) showed that kidneys from mice administered 80 mg/kg MTX haddilation of tubules, degenerative changes in the tubules,hypercellularity in the glomerulus and atypical collection ofinterstitial cells and convoluted tubules. Kidneys from miceadministered 80 mg/kg MTX+Captisol at MTX to Captisol molar ratio 1:1,and 80 mg/kg MTX+Captisol at MTX to Captisol molar ratio 1:0.5, showedmilder degeneration of tubules, normal glomerulus, and no infiltrationof inflammatory cell. However, the degree of kidney protection was muchhigher when the molar ratio of MTX to Captisol was 1:0.5.

Paraffin sections of kidney stained with hematoxylin and eosin (notshown) showed that kidneys from mice administered 120 mg/kg MTX, IV hadglomerular atrophy, degeneration of basement membrane on bowman capsule,degenerative changes in the tubules, hypercellularity in the glomerulusand infiltration of mononuclear cells. Kidneys from mice administered120 mg/kg MTX+captisol at MTX to Captisol molar ratio of 1:1, and 120mg/kg MTX+captisol at MTX to Captisol molar ratio of 1:0.5, showedmostly normal glomerulus, protection of proximal and distal tubules andno infiltration of inflammatory cells.

Example 4 Captisol MTX Time Studies

MTX—Captisol mixtures were administered as set forth in Table 4.

TABLE 4 Treatment Single intravenous bolus injection via MTX:CaptisolNumber of tail vein Tissue collection molar ratio mice Normal Mice MTX160 mg/kg Kidney collected 1:0 4 MTX 160 mg/kg + at 24 hrs after 1:1 4captisol 764 mg/kg drug MTX 160 mg/kg + administration   1:0.5 4captisol 382 mg/kg MTX 160 mg/kg +   1:0.25 4 captisol 191 mg/kg NormalMice MTX 160 mg/kg Kidney collected 1:0 4 MTX 160 mg/kg + at 48 hrsafter 1:1 4 captisol 764 mg/kg drug MTX 160 mg/kg + administration  1:0.5 4 captisol 382 mg/kg MTX 160 mg/kg +   1:0.25 4 captisol 191mg/kg Normal Mice MTX 160 mg/kg Kidney collected 1:1 4 MTX 160 mg/kg +after 1 week 1:1 4 captisol 764 mg/kg after drug administration

For light microscopic investigation, kidneys were fixed in 10% bufferedformalin and processed routinely for paraffin embedding. Tissue sectionsof 5 micron meter were stained with hematoxylin and eosine and examinedunder a Nikon coolpix light microscope, photography was done with aNikon Coolpix camera. The severity of total kidney damage was evaluatedby a semiquantitative measurement of damage as described below. Eachtissue section of the kidney was assessed for degeneration of glomerularstructure, glomerular crowding and congestion, dilation of bowman space,degeneration of proximal and distal tubules, and dilation of renaltubules, vascular congestion, and inflammatory cell infiltration. Forglomerular atrophy, glomeruli that contained more than 20 nuclei werescored “0”, and those containing less than 10 nuclei were scored “4.”Intermediate stages were 1, 2 and 3. Other criterion was scored on a 0-3scale: 0=none; 1=mild; 2=moderate; and 3=severe.

The microscopic score of each tissue was calculated as the sum of thescores given to each criterion and at least 100 nephrons (glomeruli plussurrounding tubules) were analyzed per section. See, e.g., Bhat et al.,PNAS (2003) 100(7); Sener et al., Cell Biol Toxicol (2006) 22:470-60.

Referring to FIG. 5, kidney tissue of MTX treated mice shows extensivehistopathological changes after 24 and 48 hours. At 24 hours and 48hours there was atrophy of the glomerulus, degeneration and dilation ofBowmans space, and inflammatory cell infiltration in the interstitiumand tubular degeneration. The Captisol+MTX treated group showed milderglomerular and tubular changes and less infiltration of inflammatorycells. However, some of the changes seen after 24 hours appeared to bereversible since the cumulative pathological score was less at 48 hourscompared to 24 hours.

Histopathological changes in the kidney were studied at 24 hours postMTX injection with or without captisol coadministration. Mice weresacrificed after 24 hours following MTX or MTX+Captisolcoadministration. Paraffin sections of kidney were stained withhematoxylin and eosin (not shown). MTX 160 mg/kg administration resultedin degeneration of glomerular structure and dilation of Bowman's space,degeneration of proximal and distal tubules and inflammatory cellinfiltration. Milder glomerular and tubular denegation was observed forMTX 160 mg/kg+Captisol at MTX to Captisol molar ratio of 1:1. MTX 160mg/kg+Captisol at MTX to Captisol molar ratio of 1:0.5 were mosteffective in the preservation of glomerular and tubular structures.Infiltration of inflammatory cells was completely absent. MTX 160mg/kg+Captisol used in molar ratio of 1:0.25 did not protect againstkidney damage.

Histopathological changes were recorded for the kidney at 48 hours postMTX injection with or without Captisol coadministration. Mice weresacrificed after 48 hours following MTX or MTX+Captisol administration.Paraffin sections of kidney were stained with hematoxylin and eosin (notshown). At 48 hours post injection, MTX 160 mg/kg administration (i.v.)resulted in degeneration of glomerular structure, dilation of Bowman'sspace, dilation of proximal and distal tubules, degeneration of proximaland distal tubules and inflammatory cell infiltration. Milder glomerularand tubular denegation was observed for MTX 160 mg/kg+Captisol (MTX toCaptisol molar ratio of 1:1). MTX 160 mg/kg+Captisol in 1:0.5 molarratio resulted in relatively better preservation of glomerular andtubular structure.

Infiltration of inflammatory cells was completely absent. MTX 160mg/kg+Captisol used in molar ratio of 1:0.25 were not effective inprotecting the kidney from MTX induced damage. The greatest protectionwas seen when MTX to Captisol molar ratio was 1:0.5.

Histopathological changes were also studied for the kidney after 1 weekpost MTX administration. Mice were sacrificed after 1 week following MTX160 mg/kg, single i.v. bolus injection. Paraffin sections of kidney werestained with hematoxylin and eosin (not shown). MTX administration aloneresulted mainly in degeneration of proximal and distal tubules withoccasional glomerular atrophy or crowding. Degenerating cells withswollen nuclei were seen lining the proximal and distal tubules.Occasionally some tubules were seen to be lined by double cell layer.Some tubules were congested with eosinophilic materials. Thepathological changes were mostly found in the cortical areas of thekidney. Most of the glomerular structures were normal.

Histopathological changes were recorded for the kidney after 1 week postMTX+Captisol injection. Mice were sacrificed after 1 week followingMTX+Captisol coadministration. Paraffin sections of kidney were stainedwith hematoxylin and eosin from four separate mice (not shown). Kidneysof mice coadministered with MTX 160 mg/kg+Captisol at MTX to Captisolmolar ratio of 1:1 showed significantly less glomerular disruption andgreater preservation of the tubules. Infiltration of inflammatory cellswas absent.

Example 5 Evaluation of the Nephroprotective Effect of Captisol inDoxorubicin Induced Nephrotoxicity Model in Mice

Female C57BL/6 mice were injected intravenously with a single dose of(10 mg/kg) doxorubicin. The mice were sacrificed after 72 hours. Thedevelopment of glomerular and tubulointerstitial injury afterdoxorubicin and doxorubicin+Captisol was evaluated by means of renalhistology. Paraffin sections of 5 μM were cut and stained with H&E andperiodic acid Schiff (PAS). They were examined by light microscopy andscored in a blinded fashion. Thirty glomeruli and neighboring tubuleswere scored at superficial cortex (near the surface of the capsule). Onehundred glomeruli and neighboring tubules were scored at the level ofdeep renal cortex and around the outer strips of the outer medulla.

FIG. 6 shows the mean pathology scores for each treatment group at thelevel of superficial renal cortex. FIG. 7 shows the renal pathologyscores for individual mice treated with doxorubicin ordoxorubicin+Captisol at the level of superficial renal cortex. FIG. 8shows the mean pathology scores for each treatment group at the level ofdeep renal cortex+outer medulla. FIG. 9 shows the renal pathology scoresfor individual mice treated with doxorubicin or doxorubicin+Captisol atthe level of deep renal cortex+outer medulla.

None of the control or Captisol treated mice had any tubulointerstitialchanges. The doxorubicin treated group showed tubular casts, abundantdilated tubules and moderate loss of brush border in the some proximaltubules. Some of the glomeruli were collapsed and at various stages ofdegeneration. The pathology was found to be more prominent in the outerperiphery of the renal cortex. Tubular atrophy or neutrophilsinfiltration was not seen. In doxorubicin+captisol treated mice therewas almost 71% reduction of degeneration and almost 72% reduction intubular dilatation. At deeper renal cortex and medulla levels, there wasa 90% reduction in glomerular degeneration and a 50% reduction intubular dilatation.

Example 6 Evaluation of the Nephroprotective Effect of Captisol inCisplatin Induced Nephrotoxicity Model in Mice

Female C57BL/6 mice were injected intravenously with a single dose of(10 mg/kg) cisplatin (N=5) or cisplatin+Captisol at cisplatin toCaptisol molar ratio of 1:1, 1:0.5 and 1:0.25 (N=5, N=4 and N=6,respectively). The animals were sacrificed after 72 hours.

The development of glomerular and tubulointerstitial injury aftercisplatin and protection by cisplatin+Captisol was evaluated by means ofrenal histology. Paraffin sections of 5 μM were cut and stained with H&Eand periodic acid Schiff (PAS). They were examined by light microscopyand scored in a blinded fashion. Thirty glomeruli and neighboringtubules were scored at the superficial cortex (near the surface of thecapsule). One hundred glomeruli and neighboring tubules were scored atthe level of deep renal cortex and around the outer strips of the outermedulla.

FIG. 10 shows the mean scores at the level of the superficial cortex incisplatin and cisplatin+Captisol treated groups (cisplatin to Captisolmolar ratio 1:1, 1:0.5 and 1:0.25).

FIG. 11 shows the pathology scores of individual mice in each treatmentgroup at the level of the superficial renal cortex. FIG. 12 shows themean scores at the level of the deep cortex and outer medulla incisplatin and cisplatin+Captisol treated groups (cisplatin to Captisolmolar ratio 1:1, 1:0.5 and 1:0.25). FIG. 13 shows pathology scores ofindividual mice in each treatment group at the level of the deep renalcortex and outer medulla.

None of the control mice had any tubulointerstitial changes. Thecisplatin treated mice showed necrosis, sloughing of tubular epithelialcells and loss of brush border in the some proximal tubules. Abundantpresence of dilated tubules was a prominent feature of cisplatin inducednephrotoxicity. Some of the glomeruli were collapsed and some showedearly degenerative changes. All of these changes were significantly lesspronounced by treatment with cisplatin+Captisol, demonstrating thatCaptisol protected the kidney at both cisplatin:Captisol 1:1 andcisplatin:Captisol 1:0.5.

Example 7 Nephroprotective Effect of SBE-βCD in Gentamicin InducedNephrotoxicity in Mice

Earlier studies have shown that gentamicin administration at clinicallyrelevant doses failed to produce measurable toxicity in mice. Thesedoses however, produce nephrotoxicity in humans. Therefore,nephroprotective effects of SBE-βCD in mildly renal compromised micewere evaluated. These mice were shown to exhibit pathological changes inthe kidney in clinically relevant doses. In order to produce mildlyrenal compromised mice, animals were injected with L-NAME (L argininemethyl ester) 10 mg/kg and Indomethacin 10 mg/kg, ip., 15-20 min beforethe first injection of gentamicin. Female C57BL/6 mice were administeredbolus intravenous injections for ten consecutive days with a dose of 4.0mg/kg gentamicin (N=4) or gentamicin 4.0 mg/kg+SBE-βCD 19.2 mg/kg (N=4),or gentamicin 4.0 mg/kg+SBE-βCD 38.4 mg/kg (N=4). Gentamicin to SBE-βCDmolar ratios were 1:1 and 1:2 respectively. Animals were sacrificedafter 48 hrs after the last injection. Renal pathology was assessed byevaluating H&E sections of the kidney under light microscope.Experimental details are given in Table 5 below.

TABLE 5 Groups Protocol Days of injection G:C molar ratio C57BL6 Mice(female) Gentamicin 4.0 mg/kg, 10 consecutive Without SBE-βCD N = 5administered iv. via tail days. Mice were Mildly renal vein. Bolusinjections sacrificed 72 hrs compromised mice were made. later. C57BL6Mice (female) Gentamicin 4.0 mg/kg, 10 consecutive Without SBE-βCD N = 2administered iv. via tail days. Mice were Healthy mice (not renal vein.Bolus injections sacrificed 72 hrs compromised) were made. later C57BL6Mice (female) Gentamicin 4.0 mg/kg + 10 consecutive Gentamicin:SBE- N =4 SBE-βCD 19.22 days. Mice were βCD molar ratio Mildly renal mg/kgtogether, sacrificed 72 hrs 1:1 compromised mice administered later.concurrently, iv. via tail vein. Bolus injection. C57BL6 Mice (female)Gentamicin 4.0 mg/kg + 10 consecutive Gentamicin:SBE- N = 4 SBE-βCD 38.4days. Mice were βCD molar ratio Mildly renal mg/kg, concurrently,sacrificed 72 hrs 1:2 compromised mice administered iv. later.

Paraffin sections of 5 μM were cut and stained with H&E and periodicacid Schiff (PAS). They were examined by light microscopy and scored ina blinded fashion. Five arbitrary fields at 40× magnification in eachsections was assessed for the below mentioned parameters and scoredaccording to the criteria mentioned

Dilated Tubules

0=Normal, no dilated tubules

1=mild, 1-2 dilated tubules

2=moderate, 3-5 dilated tubules

3=Severe, 6-8 dilated tubules

4=extremely severe, more than 8 dilated tubules

Tubular Casts

0=casts absent

1=mild, 1-2 casts per field

2=moderate, 3-5 casts per field

3=severe, 6-8 casts per field

4=extreme, more than 8 casts per field

Vacuoles

0=Normal, no vacuoles

1=mild, 1-4 vacuoles

2=moderate, 5-8 vacuoles

3=Severe, 9-12 vacuoles

4=extremely severe, 13-16 vacuoles

Tubular Degeneration

0=none,

1=mild, up to 25% of the visual field has degenerating tubules

2=moderate, up to 50% of the visual field has degenerating tubules

3=severe, up to 75% of the visual field has degenerating tubules

4=Extreme, more than 75% of the visual field has degenerating tubules

Inflammation

0=Normal, No inflammatory cells were seen

1=mild, up to 25% of the visual field covered with inflammatory cells

2=moderate, up to 50% of the visual field covered with inflammatorycells

3=Severe, up to 75% of the visual field covered with inflammatory cells

4=extremely severe, more than 75% of the visual field covered withinflammatory cells

Edema

0=none

1=mild

2=moderate

3=severe, 4=extreme

Under the present conditions, low dose gentamicin (4 mg/kg) givenintravenously induced no noticeable changes in the tubular cells in thehealthy kidney. However, when the mice were mild renal compromised,gentamicin at 4 mg/kg produced extensive tubular vacuolization andnecrosis in the renal cortex; the tubular cells were flattened andpartly discontinuous, and the lumens were widened. There was nonoticeable alteration the distal tubules, collecting ducts and theglomerulus.

The combination of gentamicin with SBE-βCD in a molar ratio of 1:1 and1:2 offered significant protection against gentamicin inducednephrotoxicity. The reduction in kidney pathology as a mean of all 6evaluation parameters was approximately 62%. There was attenuation oftubular dilation and proximal tubule vacuolization, and tubular castformation, as well as a reduction in tubular necrosis, and infiltrationof mononuclear cells. The effect of SBE-βCD was dose dependent, and the1:2 mole ratio of gentamicin to SBE-βCD was more effective than the 1:1mole ratio for most of the individual parameters.

Example 8 Nephroprotective Effect of SBEβCD in Acyclovir InducedNephrotoxicity in Mice

Earlier studies have shown that acyclovir administration at clinicallyrelevant doses failed to produce measurable toxicity in mice. Thesedoses however, produce nephrotoxicity in humans. Therefore, thenephroprotective effects of sulfobutylether β cyclodextrin (SBEβCD) inmildly renal compromised mice were evaluated. These mice were shown toexhibit pathological changes in the kidneys in clinically relevant dosesof acyclovir. In order to produce mildly renal compromised mice, animalswere injected with L-NAME (L arginine methyl ester) 10 mg/kg andIndomethacin 10 mg/kg, ip., 15-20 min before the first injection ofacyclovir. Female C57BL/6 mice were administered bolus intravenousinjections for ten consecutive days with a dose of 10 mg/kg acyclovir(N=3) or acyclovir 10 mg/kg+SBEβCD 173 mg/kg (N=3), or 30 mg/kgacyclovir (N=3) or acyclovir 30 mg/kg+SBEβCD 520 mg/kg (N=3) (Table 6).In both cases the acyclovir to SBEβCD molar ratio was 1:2. Animals weresacrificed after 48 hrs after the last injection. Renal pathology wasassessed by evaluating H&E sections of the kidney under a lightmicroscope.

TABLE 6 SBEβCD Acyclovir:SBEβCD Acyclovir Acyclovir dose & SBEβCD molarKidney dose/regimen [Molarity] regimen [molarity] ratio collection 10.0mg/kg, IV, for  4.0 mM 0.0  0.0 N/A 48 hrs. after the 10 consecutivedays last injection (n = 3) 10.0 mg/kg, IV, for  4.0 mM 173 mg/kg  8.0mM 1:2 48 hrs. after the 10 consecutive days [8.0 mM], last injection (n= 3) concurrently 30.0 mg/kg, IV, for 12.1 mM 0.0  0.0 N/A 48 hrs. afterthe 10 consecutive days last injection (n = 3) 30.0 mg/kg, IV, for 12.1mM 520.0 mg/kg 24.2 mM 1:2 48 hrs. after the 10 consecutive days [24.2mM], last injection (n = 3) concurrently

Paraffin sections of 5 μM were cut and stained with H&E and periodicacid Schiff (PAS). They were examined by light microscopy and scored ina blinded fashion. Five arbitrary fields at 40× magnification in eachsections was assessed according to the below mentioned parameters andscored according to the criteria mentioned.

Dilated Tubules

0=Normal, no dilated tubules

1=mild, 1-2 dilated tubules

2=moderate, 3-5 dilated tubules

3=Severe, 6-8 dilated tubules

4=extremely severe, more than 8 dilated tubules

Vacuoles

0=Normal, no vacuoles

1=mild, 1-4 vacuoles

2=moderate, 5-8 vacuoles

3=Severe, 9-12 vacuoles

4=extremely severe, 13-16 vacuoles

Inflammation

0=Normal, No inflammatory cells were seen

1=mild, up to 25% of the visual field covered with inflammatory cells

2=moderate, up to 50% of the visual field covered with inflammatorycells

3=Severe, up to 75% of the visual field covered with inflammatory cells

4=extremely severe, more than 75% of the visual field covered withinflammatory cells

Increase in the Number of Tubular Cells

0-absent

1=mild, up to 25% of the tubules have increased cell numbers oroverlapping of cells

2=moderate, up to 50% of the tubules have increase cell numbers oroverlapping of cells

3=Severe, up to 75% of the tubules have increased cell numbers oroverlapping of cells

4=extremely severe, more than 75% of the visual field covered withinflammatory cells

Chronic administration of acyclovir in mildly renal compromised micecaused a dose dependent increase in pathological changes in themorphology of the kidney. Acyclovir treatment produced moderate tosevere signs of tubulopathy as indicated by numerous dilated tubules andirregular vacuolization of the proximal tubular epithelium. Other signsof tubular damage were absent, such as microcalcification or necrosis ofepithelial cells. Mild infiltration of mononuclear cells was seen in theparenchyma. Most of the pathology, in particular tubular dilation, wasseen along the periphery of the cortex. Glomerular pathology wasminimal.

Concurrent administration of SBEβCD in a molar ratio of 1:2 resulted insignificant attenuation of tubular dilation and vacuole formation in thetubules in both the outer cortex and in the medulla. In addition, therewas attenuation in tubular casts and infiltration of mononuclear cellswithin the parenchyma. The reduction in kidney pathology as a mean ofthe several parameters evaluated was 62% for the 30 mg/kg dose ofacyclovir and 67% for the 10 mg/kg dose.

Example 9 Nephroprotective Effect of SBE-βCD in Iohexyl InducedNephrotoxicity in Mice

Iohexyl is nephrotoxic in humans and can produce acute renal failure inrenally compromised patients. Therefore, the nephroprotective effects ofsulfobutyl ethyl β cyclodextrin (SBE-βCD) in a contrast agent inducednephropathy model involving mildly renal compromised mice wereevaluated. These mice were shown to develop pathological changes in thekidney in clinically relevant doses of iohexyl. In order to producemildly renal compromised mice, female C57BL/6 mice were injected withnitric oxide synthase inhibitor L-NAME (L arginine methyl ester) 10mg/kg and prostaglandin synthesis inhibitor Indomethacin 10 mg/kg, ip.,15-20 min before injecting of iohexyl. The mice were administered asingle bolus intravenous injections of iohexyl 1.5 g/kg (N=4) or iohexyl1.5 g/kg+SBE-βCD 1.3 g/kg (N=4), or iohexyl 1.5 g/kg+SBE-βCD 1.3 mg/kgadministered 30 minutes before, concurrently, and 30 min after iohexylinjection (N=4)(Table 7). In both cases, the iohexyl to SBE-βCD molarratio was 1:0.1 during the concurrent dose. However, in the second casethe molar concentration in vivo is expected to be higher. Animals weresacrificed 24 hrs after the iohexyl injection. Renal pathology wasassessed by evaluating H&E sections of the kidney under a lightmicroscope.

TABLE 7 Iohexol Iohexol SBE-βCD dose I:SBE βCD Kidney dose/regimen[Molarity] & regimen molar ratio collection 1.5 g/kg, iv., once 423 mM0.0 0.0 24 hrs. after (n = 4) Iohexol injection 1.5 g/kg, iv., once 423mM 1.3 g/kg, [141.9 1:0.1 24 hrs. after (n = 4) mM], concurrentlyIohexol injection 1.5 g/kg, iv., once 423 mM 1.3 g/kg each, 3 1:0.1 (forthe 24 hrs. after (n = 4) doses, given 30 min concurrent Iohexol before,during, and dose) injection 30 min after Iohexol injection

Paraffin sections of 5 μM were cut and stained with H&E and periodicacid Schiff (PAS). They were examined by light microscopy and scored ina blinded fashion. At 40× magnification, five arbitrary fields in eachsection was assessed for the below mentioned parameters and scoredblindly according to the criteria mentioned. A total of 4 sections wereanalyzed per kidney.

Administration of the contrast agent iohexyl in mildly renal-compromisedmice caused pathological changes in the morphology of the kidneytubules. Iohexyl treatment produced moderate to severe signs oftubulopathy as indicated by the presence of numerous dilated tubules,tubular casts, vacuolization of the proximal tubular epithelium, anddegeneration of proximal tubules. Other signs of tubular damage wereabsent, such as basophilia, microcalcification or necrosis of epithelialcells. Most of the pathology, in particular tubular dilation, was seenalong the periphery of the cortex. The majority of the glomeruliappeared normal.

The concurrent administration of SBE-βCD with iohexyl to SBE-βCD at amolar ratio of 1:0.1 resulted in significant attenuation of tubulardilation, tubular cast formation, and vacuolization of the tubularepithelium in both the outer cortex and the medulla. The protectiveeffect of SBE-βCD appeared to be enhanced when it was given in 3 doses(once before, during and after iohexyl injection). However, an increasein vacuolization was seen in these mice which could be due to a highlocal concentration of SBE-βCD achieved in the mouse kidney. Higherdoses of SBE-βCD (in mice) have been shown to produce reversiblevacuolization of the proximal tubules. Even though 1.3 g/kg is belowwhat is expected to produce vacuolization in mice, the cumulative dosemay have been higher. The reduction in kidney pathology score as a meanof the several parameters evaluated was approximately 72% when theSBE-βCD was given in one dose concurrently with the iohexyl.

Example 10 Nephroprotective Effect of HPβCD in a Methotrexate InducedNephrotoxicity Model in Mice

To determine the protective effect of other cyclodextrin molecules,2-hydroxypropyl β-cyclodextrin (HPβCD) was evaluated. Female C57BL/6mice were injected intravenously with a single dose of methotrexate 80mg/kg, IV, or methotrexate+HPβCD, IV, at methotrexate to HPβCD molarratios of 1:1 and 1:0.5. The animals were sacrificed after 24 hrs. Thedevelopment of glomerular and tubulointerstitial injury aftermethotrexate and protection by methotrexate+HPβCD was evaluated by meansof assessment of renal histology by light microscopy. Leukovorin wasgiven 4 hrs and after 18 hrs. Urine was not alkalinized.

Paraffin sections of 5 μM were cut and stained with H&E and periodicacid Schiff (PAS). They were examined by light microscopy and scored ina blinded fashion. Five arbitrary fields at 40× magnification in eachsection were assessed for the parameters, and scored according to thecriteria, set forth in Example 7.

In normal mice a single IV injection with MTX at 80 mg/kg producedsignificant histopathological changes in the kidney in the form ofmildly atrophied glomeruli, tubular degeneration, cast formation,dilation and infiltration of mononuclear cells. In mice treatedconcurrently with MTX+HPβCD (with both 1:1 and 1:0.5 molar ratios) therewas significant reduction in tubular pathology and better preservationof glomerular structure was seen. Overall MTX: HPβCD molar ratio of1:0.5 was more effective than a molar ratio of 1:1. The reduction inkidney pathology as a mean of the several parameters measured wasapproximately 55%.

1. A method of reducing nephrotoxicity associated with administration ofcisplatin to a patient in need thereof, the method comprising:administering a pharmaceutical composition to the patient, thecomposition comprising cisplatin, a substituted cyclodextrin, and apharmaceutically acceptable carrier.
 2. The method of claim 1 whereinthe substituted cyclodextrin is of the formula:cyclodextrin-[(O—R—Y)⁻(Me)⁺]_(n) where R is selected from the groupconsisting of straight chained or branched C₁₋₁₀ alkyl, alkenyl oralkynyl; Y is an anionic group selected from COO, SO₃, SO₄, PO₃H or PO₄;Me is a pharmaceutically acceptable cation; and n is a whole numbergreater than
 1. 3. The method of claim 2 wherein the cyclodextrin isbeta-cyclodextrin.
 4. The method of claim 3 wherein R is C₁₋₄ alkylselected from the group consisting of methyl, ethyl, propyl and butyl.5. The method of claim 4 where R is butyl.
 6. The method of claim 2wherein Y is SO₃.
 7. The method of claim 3 wherein (O—R—Y)⁻(Me)⁺ is—O—(CH₂)₄—SO₃ ⁻Na⁺.
 8. The method of claim 7 wherein n is
 7. 9. Themethod of claim 1 wherein the substituted cyclodextrin is2-hydroxypropyl β-cyclodextrin.
 10. The method of claim 1 wherein themolar ratio of cisplatin to substituted cyclodextrin is selected from aratio in the range from 50:1 to 1:50.
 11. The method of claim 10 whereinthe molar ratio of cisplatin to substituted cyclodextrin is selectedfrom a ratio in the range from 25:1 to 1:20.
 12. The method of claim 11wherein the molar ratio of cisplatin to substituted cyclodextrin isselected from a ratio in the range from 10:1 to 1:10.
 13. The method ofclaim 12 wherein the molar ratio of cisplatin to substitutedcyclodextrin is selected from a ratio in the range from 2:1 to 1:5. 14.The method of claim 13 wherein the molar ratio of cisplatin tosubstituted cyclodextrin is selected from the group consisting of 2:1;1:1; 1:2; and 1:5.
 15. A method of reducing nephrotoxicity associatedwith administration of cisplatin to a patient in need thereof, themethod comprising: administering to the patient in need thereof a firstpharmaceutical composition comprising a substituted cyclodextrin, andadministering to the patient a second pharmaceutical compositioncomprising cisplatin.
 16. The method of claim 15 wherein theadministering of the first pharmaceutical composition is performed atany period of time selected from within one hour before to within onehour after administering the second pharmaceutical composition.
 17. Themethod of claim 16 wherein the administering of the first pharmaceuticalcomposition is performed concurrently with the administering of thesecond pharmaceutical composition.
 18. The method of claim 15 whereinsaid substituted cyclodextrin is a cyclodextrin of the formula:cyclodextrin-[(O—R—Y)⁻(Me)⁺]_(n) where R is selected from the groupconsisting of straight chained or branched C₁₋₁₀ alkyl, alkenyl oralkynyl; Y is an anionic group selected from COO, SO₃, SO₄, PO₃H or PO₄;Me is a pharmaceutically acceptable cation; and n is a whole numbergreater than
 1. 19. The method of claim 18 wherein said cyclodextrin isβ-cyclodextrin; and (O—R—Y)⁻(Me)⁺ is —O—(CH₂)₄—SO₃ ⁻Na⁺.
 20. The methodof claim 15 wherein said cyclodextrin is 2-hydroxypropyl β-cyclodextrin.21. The method of claim 15 wherein the molar ratio of cisplatin in thesecond composition to substituted cyclodextrin in the first compositionis selected from a ratio in the range from 50:1 to 1:50.
 22. The methodof claim 21 wherein the molar ratio of cisplatin in the secondcomposition to substituted cyclodextrin in the first composition isselected from a ratio in the range of 25:1 to 1:20.
 23. The method ofclaim 22 wherein the molar ratio of cisplatin in the second compositionto substituted cyclodextrin in the first composition is selected from aratio in the range from 10:1 to 1:10.
 24. The method of claim 23 whereinthe molar ratio of cisplatin in the second composition to substitutedcyclodextrin in the first composition is selected from a ratio in therange from 2:1 to 1:5.
 25. The method of claim 24 wherein the molarratio of cisplatin in the second composition to substituted cyclodextrinin the first composition is selected from the group consisting of 2:1;1:1; 1:2; and 1:5.
 26. The method of claim 1 wherein the pharmaceuticalcomposition is administered intravenously.
 27. The method of claim 15wherein the first pharmaceutical composition and the secondpharmaceutical composition are both administered intravenously.